Mechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism

Horne C, Venugopal H, Panjikar S, Henrickson A, Brookes E, North R, Murphy J, Friemann R, Griffin M, Ramm G, Demeler B, Dobson R, (2020) DOI

SASDHT9 – Dimeric NanR-DNA hetero-complex from Escherichia coli

HTH-type transcriptional repressor NanR
(GGTATA)2 repeat DNA
MWexperimental 66 kDa
MWexpected 70 kDa
VPorod 108 nm3
log I(s) 1.04×10-1 1.04×10-2 1.04×10-3 1.04×10-4
HTH-type transcriptional repressor NanR (GGTATA)2 repeat DNA small angle scattering data  s, nm-1
ln I(s)
HTH-type transcriptional repressor NanR (GGTATA)2 repeat DNA Guinier plot ln 1.04×10-1 Rg: 3.3 nm 0 (3.3 nm)-2 s2
HTH-type transcriptional repressor NanR (GGTATA)2 repeat DNA Kratky plot 1.104 0 3 sRg
HTH-type transcriptional repressor NanR (GGTATA)2 repeat DNA pair distance distribution function Rg: 3.3 nm 0 Dmax: 9.8 nm

Data validation

Fits and models

log I(s)
 s, nm-1
HTH-type transcriptional repressor NanR (GGTATA)2 repeat DNA CUSTOM IN-HOUSE model

NanR-DNA hetero-complex was prepared by incubating NanR (340 µM) and DNA with two GGTATA repeats (170 µM) on ice for 30 min prior to injection. Samples were prepared in 20 mM Tris-Hcl (pH 8.0), 150 mM NaCl, 0.1 % (w/v) sodium azide, pH 8 and were collected using co-flow size exclusion chromatography SAXS (SEC-SAXS) on the SAXS/WAXS beam line at the Australian Synchrotron (Melbourne, Australia) using a Pilatus 1M detector at a sample-detector distance of 1.6 m and at a wavelength of λ = 0.10332 nm (l(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). Co-flow SEC-SAXS was performed at 12°C using the following parameters: Column: Superdex S200 Increase 5/150 GL (GE Healthcare); Flow rate: 0.45 mL/min; Injection volume: 70 μL. The data were collected through the SEC peak of the protein as a series of 1 second exposures. Each unsubtracted data frame was normalised to the intensity of the transmitted beam and radially averaged using the Scatterbrain software package (Australian Synchrotron). The scattering of an appropriate solvent-blank was subtracted. The resulting subtracted frames were scaled and averaged to generate the final SAXS profile displayed in this entry.

HTH-type transcriptional repressor NanR (NanR)
Mol. type   Protein
Organism   Escherichia coli
Olig. state   Dimer
Mon. MW   29.5 kDa
UniProt   P0A8W0 (1-263)
Sequence   FASTA
(GGTATA)2 repeat DNA
Mol. type   DNA
Olig. state   Monomer
Mon. MW   10.5 kDa
Sequence   FASTA